PROJECT SUMMARY Pseudomonas aeruginosa (PA) is the leading causative agent in microbial keratitis. During contact lens wear, host defense mechanisms are compromised. This allows PA to breach the tight corneal barrier and infect the otherwise healthy eye. Recently, exosomes have been implicated as major players in inflammation and infection. In addition, exosomes are becoming increasingly recognized as potential therapeutic agents. Our preliminary data shows that there is massive exosome release from corneal epithelial cells during infection by PA. These exosomes contain a unique mixture of proteases, transcriptional regulators and proteins involved in immune regulation. We further provide data that indicates that these exosomes both promote neutrophil activation and convey protection to corneal epithelial cells against further invasion. Moreover, we have found that exosomes isolated from autologous body fluids have antimicrobial and immunomodulatory properties. Based on our findings, we propose the primary hypothesis that exosomes released from PA-infected corneal epithelial cells promote PA clearance by innate immune cells and prime non-infected corneal epithelial cells to defend against PA infection. We further propose the secondary hypothesis that exosomes derived from exosome rich body fluids contain potent antimicrobial and anti-inflammatory mediators that can be harnessed to promote PA clearance and disease resolution in the cornea. We will test these hypotheses as follows: Aim 1. Establish how exosomes isolated from PA-infected corneal epithelial cells and human body fluids impact innate immune cells in vitro. Aim 2. Determine how exosomes isolated from PA-infected corneal epithelial cells and human body fluids impact the corneal epithelial response to PA in vitro. Aim 3. Determine whether exosomes isolated from autologous human body fluids exhibit protective antimicrobial and immunomodulatory properties in the rabbit contact lens model in vivo. To accomplish these studies, we have compiled a highly collaborative multidisciplinary team and have access to state of the art resources in the Department of Ophthalmology and UT Southwestern core facilities. These studies are significant and innovative because they are the first of their kind for PA infection in any tissue or cell system. The potential therapeutic use of exosomes from human body fluids represents a major paradigm shift for treating corneal infections and has broad therapeutic implications.